TY - JOUR
T1 - A rapid method for reconstitution of bacterial membrane proteins
AU - Varadhachary, A.
AU - Maloney, P. C.
PY - 1990/8
Y1 - 1990/8
N2 - We have devised a simple method for the reconstitution of bacterial membrane proteins directly from small (1–20 ml) volumes of cell culture, thus eliminating the preparation of membrane vesicles. Cells are subjected to simultaneous lysozyme digestion and osmotic lysis, and after brief centrifugation ghosts are solubilized in 1.2% octyl‐β‐D‐glucopyranoside (octyl‐glucoside) in the presence of added carrier lipid and an osmolyte. Aliquots of the clarified supernatant are suitable for reconstitution, as documented by using extracts from three different Gram‐negative cells to recover both inorganic phosphate (Pi)‐linked antiport and oxalate: formate exchange activities in proteoliposomes. These proteoliposomes are physically stable, non‐leaky and can sustain a membrane potential and, because functional porins do not reconstitute, the artificial system has transport characteristics similar to those found when proteoliposomes are obtained using standard methods. This method should become an important tool for the screening and characterization of large numbers of strains, both wild‐type and mutant.
AB - We have devised a simple method for the reconstitution of bacterial membrane proteins directly from small (1–20 ml) volumes of cell culture, thus eliminating the preparation of membrane vesicles. Cells are subjected to simultaneous lysozyme digestion and osmotic lysis, and after brief centrifugation ghosts are solubilized in 1.2% octyl‐β‐D‐glucopyranoside (octyl‐glucoside) in the presence of added carrier lipid and an osmolyte. Aliquots of the clarified supernatant are suitable for reconstitution, as documented by using extracts from three different Gram‐negative cells to recover both inorganic phosphate (Pi)‐linked antiport and oxalate: formate exchange activities in proteoliposomes. These proteoliposomes are physically stable, non‐leaky and can sustain a membrane potential and, because functional porins do not reconstitute, the artificial system has transport characteristics similar to those found when proteoliposomes are obtained using standard methods. This method should become an important tool for the screening and characterization of large numbers of strains, both wild‐type and mutant.
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U2 - 10.1111/j.1365-2958.1990.tb00720.x
DO - 10.1111/j.1365-2958.1990.tb00720.x
M3 - Article
C2 - 2280690
AN - SCOPUS:0025110961
SN - 0950-382X
VL - 4
SP - 1407
EP - 1411
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 8
ER -